The use of GHB/GBL to tamper drinks and perform
unlawful acts is an increasing problem in today’s society. In alleged “date
rape” cases, it is the job of a forensic scientist to determine if foul play has
occurred. Forensic scientists must tackle many challenges in order to obtain the
truth. One example of this challenge is the fact that GBL naturally occurs in
wine. Once ingested, GBL readily converts to GHB, causing many side effects.
This report tackles the question, “Do products such as wine create a false
positive in forensic testing?” Using the ultra-violet spectrophotometer, a
standard curve was created using the standard concentrations of GBL. This
standard curve was used to determine the concentrations of GBL in four different
kinds of wines after extraction. The results confirmed significant levels of GBL
in wine, suggesting that an analysis of human consumption of alcohol needs to be
more thoroughly explored in order to completely rule out the possibility of wine
creating a false positive test when evaluating alleged date rape cases.

Introduction:

The name GHB, gamma-hydroxybutyrate, brings to
mind the compound used in “date rape” cases. In fact, it is the cause of many of
these unfortunate events; however, it can be used for alternative purposes as
well. GHB has been around since the 1960’s, when it was used as an anesthetic.
Since then, it has been used for treating insomnia, alcoholism, cerebrovascular
disorders (Kintz, 2003), and narcolepsy (Ciolino et al., 2001). Unfortunately,
the drug had been so badly abused that in 2000, the United States government had
to label GHB as a DEA, Drug Enforcement Administration, Control I substance
(Hennessy, 2004).

This drug is most often used by rapists in
clubs and bars. It is often found that women have their alcoholic drink spiked
by GHB. Once women have been drugged, they usually report symptoms such as
drowsiness, confusion, dizziness, impaired memory, impaired motor skills, and a
paralysis-like state. It also promotes the hormones in the body that aid in
arousal (Schwartz, 2000). Bodybuilders also abuse GHB as a hormone booster.
However, the effects of GHB for the purpose of bodybuilding are highly debated
(Chappell, 2004).

A compound which is closely related to GHB is
GBL, Gamma-Butyrolactone. GBL is a lactone of GHB, which is a hydroxylated short
chain carboxylic acid. GBL is converted to GHB by hydrolysis, and the reverse
reaction is caused by intramolecular esterification (Ciolino et al., 2001).

GBL
GHB

GHB is naturally occurring in the body and closely
resembles the neurotransmitter gamma-aminobutyric acid (GABA). In mammalian
bodies, GABA is converted into GHB. GHB is also formed naturally in the body
with the ingestion of GBL. The structures of GHB, GBL, and GAMA are shown in
Chart I.

Chart I:

Structures of
GHB, GBL, and GAMA

GHB

GBL

GAMA

GBL is listed only as a List I
chemical in the United States. This presents a problem because GBL is present in
many commercial products causing the misuse of these products (Ciolino et al.,
2001). GBL also naturally occurs in wine, in which it is produced during the
fermentation process (Vose, 2001). Taking these factors into consideration, a
number of tests have been developed to test for GBL/GHB ratio. Since forensic
scientists must consider this ratio before evaluating a rape case or any other
criminal case related to GHB.

Factors that affect the rate of conversion of
GBL to GHB are the pH level, time, and temperature. According to the work of
Ciolino et al., 2001, at acidic pH’s around 2.0, a 2:1 equilibrium ratio of
GBL:GHB occurs over a period of nine days (analyzed by HPLC/MS). At basic pH
levels, especially around 12.0, an almost complete conversion of GBL to GHB
happens within minutes. At the pH levels of 4.0 to 7.0, the conversion process
takes several months. Also, at high temperatures, the reaction is significantly
faster compared to that at low temperatures (Ciolino et al., 2001).

Forensic science has made significant advancements in the
area of alleged date rape cases, including the effects of pH level, time, and
temperature on the conversion rate of GBL to GHB. However, there are still
unanswered questions within different fields of investigation on this subject.
This study is an attempt to answer some of these questions.

An earlier study showed that the analysis of grape juice
does not indicate the presence of GBL (Vose, 2001). Wine, which is composed
entirely of grape juice and alcohol, has also been analyzed to determine its GBL
content and analysis confirms that GBL is present in wine. This indicates that
GBL is produced during the fermentation process used to make wine (Vose, 2001).
During the fermentation of wine, sugar is converted to alcohol using yeast. This
reaction yields ethanol, with GBL and carbon dioxide as by-products (Ough,
1987). Many different kinds of wines contain GBL, which once ingested, converts
to GHB. Testing wine samples using a color indicator test and IR spectroscopy
determines the presence of GBL in certain wines (Chappell, 2004). GBL gives a
unique IR spectrum that is easy to determine and analyze (Chappell, 2004):

Chart I:

IR spectrum of
GBL

Chart II:

Analysis of GBL IR spectrum

Vibrational Mode

Wavenumber

(cm-1)

Strength of Peak

Carbonyl stretching mode

1770

Very
strong

OH
bond stretching

3525

Relatively weak

C-H
bond

2992

Relatively weak

In
plane deformation of the carboxyl hydrogen and stretching of the
carbon oxygen bond in the carboxyl group

1450-1150

Strong

Stretching C-O bond of the terminal hydroxyl group

1038

Strong

(Harvey, 2000)

The color indicator test (Morris)
is another qualitative test that indicates the presence of GBL within the
sample. If GBL is present in the sample, formation of a purple color appears.
The higher the GBL content in the sample, the darker the purple color. This is a
quick and efficient testing method for GBL that many forensic scientists employ,
but it is not a quantitative method.

In this study, a quantitative
analysis technique using ultra-violet spectrophotometer is used in order to
determine the concentration of GBL in several different kinds of wine. The π to
π* electron transition of the carbonyl (C=O) functional group in GBL absorbs in
the ultra-violet region, which can be used for quantitative testing. Results of
this study prove that wine contains enough GBL to actively affect a forensic
science drug test. Since GBL and GHB are so closely related, it is important to
determine their ratio in the equilibrium mixture at different pH levels. At a pH
of 12, the hydrolysis of GBL to GHB is a complete conversion within fifteen
minutes (Ciolino et al.., 2001). Between a pH of 12 and a pH of 3, the
equilibrium takes several months and the equilibrium concentration of GBL
increases as pH decreases. However, at a pH of 2, the equilibrium reaction takes
nine days to reach 68% GBL and 32% GHB in the mixture (Ciolino et al., 2001).
The average soda has a pH of 2.8 and most drinks tainted with GBL are alcoholic
soda drinks. Therefore, the GBL-GHB equilibrium study is conducted at a pH of 2
using a phosphate buffer. The absorbance is recorded initially every hour for
six hours and then every day for nine days at the wavelength of maximum
absorption, since the equilibrium reaction is logarithmic and will occur more
rapidly in the beginning hours than over the total nine-day equilibrium process.

The IR spectrum in Chart III allows identification of
certain compounds within wine. The broad peak from 3600 – 3000 cm-1
is due to the alcohol in the wine. The smaller, semi-wide peak just below 1800
due to the carbonyl stretching represents the key identifier of GBL. However,
the IR spectroscopy cannot conclude any quantitative data useful for determining
concentration of GBL in wine. Other attempts utilizing the visible absorbance
spectrophotometer also concluded that the color indicator test was a useful
qualitative analysis technique; however, it could not be used for quantitative
analysis.

II. Standardization of GBL using
UV Absorption:

1 x 10-3 M standard GBL solution was scanned
from 200nm to 300nm in order to find the optimum wavelength of absorbance for
testing.

Graph I:

Determination
of wavelength of maximum absorption

The optimum absorbance was found at 205nm. A
standard curve is obtained using standard solutions of GBL at a range of
concentrations (1x10-3M to 1x10-4M) and measuring their
absorbance at the wavelength of maximum absorption.

Chart IV:

Absorbance of
standard solutions

1 x 10-3
to 1 x 10-4 M GBL at 205 nm

Concentration
(M)

Absorbance

1.00x10-3

0.551

9.00x10-4

0.500

8.00x10-4

0.436

7.00x10-4

0.385

6.00x10-4

0.320

5.00x10-4

0.277

4.00x10-4

0.230

3.00x10-4

0.231

2.00x10-4

0.178

1.00x10-4

0.099

Graph II:

Standard curve

1x10-3
M to 1x10-4 M GBL at 205 nm

III.Quantification of GBL in wine samples using UV absorption:

Once a standard curve is
established, the concentration of GBL in wine was determined. The wine samples
are prepared using chloroform to extract GBL from wine by mixing 10µl of wine
sample with 10µl of chloroform in a separator funnel. Then GBL is extracted to a
water phase from chloroform using multiple extractions. GBL in aqueous phase is
used for absorption measurements.

Each sample tested, however,
needed to be diluted differently depending upon amount of GBL in solution. The
samples of wine tested were from Italy, Germany, California (white), and
California (red).

Chart V:

Wine samples

Wine

Dilution ratio

Absorbance 1

Absorbance 2

Average
absorbance

California (red)

1:40

0.841

0.789

0.815

California
(white)

1:20

0.874

0.801

0.838

Germany

1:30

0.636

0.677

0.657

Italy

1:20

0.465

0.477

0.471

IV.Kinetics of GBL to GHB conversion:

The rate of conversion of GBL to GHB is
measured at a pH of 2. Using a phosphoric acid buffer, a nine-day kinetics study
was conducted.

Chart VI:

Absorbances in time
(first six hours) at pH 2

at three
different GBL concentrations

time (hr)

1.00x10-4
M

5.00x10-4
M

1.00x10-3
M

0

0.048

0.247

0.519

1

0.046

0.241

0.501

2

0.045

0.239

0.481

3

0.042

0.234

0.469

4

0.041

0.225

0.451

5

0.040

0.211

0.437

6

0.039

0.201

0.423

Graph III:

Absorbances
vs. time (first six hours) at pH 2

at three
different GBL concentrations

Chart VII:

Absorbances
vs. time (nine days) at pH 2

at three
different GBL concentrations

Time (days)

1.00x10-4
M

5.00x10-4
M

1.00x10-3
M

2

0.039

0.199

0.419

3

0.037

0.193

0.410

4

0.037

0.186

0.389

5

0.036

0.177

0.377

6

0.035

0.175

0.362

7

0.035

0.173

0.351

8

0.034

0.171

0.349

9

0.034

0.172

0.348

Graph IV:

Absorbances
vs. time (nine days) at pH 2

at three
different GBL concentrations

Data analysis and results:

I.GBL concentrations in wine samples:

Using the standard curve equation and relative
absorbance depending upon dilution, the concentration of GBL in wine was
experimentally determined.

Chart VIII:

Experimentally
determined GBL concentrations in wine samples

Wine type

Absorbance

Concentration, M
(dilution ratio)

Original
concentration, M

California (red)

0.815

1.588x10-3(1:40)

6.352 x 10-3

California (white)

0.838

1.636x10-3
(1:20)

3.272 x 10-3

Germany

0.657

1.255x10-3
(1:30)

3.765 x 10-3

Italy

0.471

0.866x10-3
(1:20)

1.732 x 10-3

II.Kinetics of GBL to GHB conversion:

The six hour study at a pH of 2
resulted in a first order relation between ln absorbance and time.

Chart IX:

ln absorbance
vs. time (six hour study) at pH 2 at three different concentrations

time (hr)

1 x 10-4
M

5 x 10-4
M

1 x 10-3
M

0

-3.037

-1.398

-0.656

1

-3.079

-1.423

-0.691

2

-3.101

-1.431

-0.732

3

-3.170

-1.452

-0.757

4

-3.194

-1.492

-0.796

5

-3.219

-1.556

-0.828

6

-3.244

-1.604

-0.860

Graph V:

ln absorbance
vs. time (six hour study) at pH 2

at three
different concentrations

The rate constant of GBL to GHB conversion reaction can be
determined using Graph V since k = - slope.

Chart X:

Rate constants
measured at three different concentrations

Concentration (M)

Experimental k value (hr -1)

1.00x10-4

.0340

5.00x10-4

.0337

1.00x10-3

.0344

average k =
.034433 hr -1

III. Equilibrium amounts of GBL and GHB:

The equilibrium ratio of the
mixture is determined by finding the ratio of initial absorbance to the final
absorbance.

Chart XI:

Equilibrium
ratio of GBL:GHB

Concentration

1.00x10-4 M

5.00x10-4 M

1.00x10-3 M

Absorbance after nine days

.0340

.1720

.3480

Initial absorbance

.0480

.2470

.5190

Ratio
of GBL:GHB

.7083

.6964

.6705

Chart XII:

Equilibrium
percent GBL:GHB

concentration

1.00x10-4 M

5.00x10-4 M

1.00x10-3 M

Percent GBL (%)

70.83

69.64

67.05

Percent GHB (%)

29.17

30.36

32.95

Average Percent
GBL = 69.2%

Average
percent GHB = 30.8%

Conclusion and Discussion:

The purpose of this study was to determine if
wine contains enough GBL to create a “false positive” during a forensic drug
test. To answer this question, different analysis techniques were tested to
determine the concentration of GBL in wine. IR spectrum was able to verify GBL’s
presence in wine; however, it failed to determine the concentration levels.
Further investigation of the ultra-violet region led to the detection of the
carbonyl group’s pi electron excitation which has a maximum absorption around
205nm. A standard curve using pure standard GBL solutions was established at
this wavelength. After testing the wine samples, the standard curve was used to
determine the concentration of GBL. The concentrations of GBL in wine samples
are as follows:

Chart XIII:

Concentration
of GBL in wine

Wine

Concentration
(M)

California (red)

6.352 x 10-3

California
(white)

3.272 x 10-3

Germany (white)

3.765 x 10-3

Italy (white)

1.732 x 10-3

The results show that the
California red wine contains the highest concentration of GBL, while the Italian
white wine contains the lowest concentration. These results are consistent with
those of earlier study (Ciolino et al., 2001) in that red wine has higher levels
of GBL than white wine and that the Italian wine contains the lowest
concentration of GBL.

These concentrations suggest that enough
consumption of wine could affect a forensic drug test. However, the results of
this study are not conclusive enough to verify this theory. A further analysis
should be conducted involving human consumption to confirm this thesis. The
results of this experiment suggest that, it is overwhelmingly possible that the
consumption of wine could affect a forensic drug test.

Forensic science deals with
GHB/GBL in alleged date rape cases. When a victim makes claims of being raped
and/or drugged, the police department’s forensic scientists attempt to find
scientific evidence supporting or eliminating the subject’s allegations.
Depending on the case, different techniques are employed to find this evidence.
The easiest determination of GHB is to analyze the drink that was claimed to be
tainted. However, in most cases, the victim is the most reliable object to base
judgment upon. Blood, urine, and hair testing can also be used to determine GHB;
however, these tests must be conducted in a timely manner due to the fact that
GHB only exits in the body for a short time. Another factor to be considered is
that GHB naturally occurs within the body. During normal brain metabolism, GABA
(gamma-aminobutyric acid) is converted to GHB (Vose, 2001). The natural amounts
of GHB within the body are below a forensic drug testing threshold. However,
when outside or artificial sources of GHB (such as wine) ingested additionally,
the level of GHB maybe increased to levels above the forensic testing
threshold.

This thesis and the reasons
stated above call for another study to be conducted to account for GHB naturally
occurring in the body as well as the GHB amount due to the ingestion of products
containing GBL (i.e. wine). The threshold of the color indicator test is 3.00x10-3
M, which is fairly close to the GBL concentrations found in wine. All forensic
tests using blood, urine, and hair have a threshold limit relatively close to
the concentration of GBL in wine. Therefore, a further study testing subjects
before and after ingesting wine to determine if GHB levels rise to create a
“false positive” would be more conclusive. If consumption of wine affects the
GHB test, then forensic scientists must develop a new test to eliminate the
interferent’s affect within the testing procedure.

The forensics lab, when dealing with alleged
rape cases, focuses its investigation towards tainted drinks. Since GHB is a
Control I substance, GBL is much easier to obtain and often replaces GHB due to
the conversion between the two. Most drinks tainted by GBL are mixed drinks that
contain alcohol and dark soda (however, anything can be tainted due to the
colorless and almost odorless character of GBL). The average soda has a pH of
2.8. Therefore, the study of equilibrium ratio was conducted at a pH of 2.
According to Chappell, at a pH of 2, the hydrolysis of GBL to GHB is a nine day
process and equilibrates to 68%GBL/32%GHB. The percent equilibrium found in this
study was 69.2% and 30.8%, respectively. The agreement between the earlier study
and our data is within 2.7%. This close agreement concludes that this experiment
using the ultra-violet spectrophotometer was highly accurate and precise
compared to previous studies done with HPLC/MS. The rate constant of GBL to GHB
conversion is found to be .034433/hr. This rate constant is an extremely useful
quantitative characteristic of the chemical conversion between GBL and GHB. All
data obtained in this study is in agreement with previous studies done; however,
this is the first known study using the ultra-violet spectrophotometer.